The single most polymorphic STR Locus: SE33 performance in U.S. populations
Received 25 August 2009; accepted 27 August 2009. published online 15 October 2009.
Abstract
The STR locus SE33 (ACTBP2) located on chromosome 6 (6q14) is arguably the most polymorphic marker examined thus far by the forensic community with a heterozygosity of >0.95 in some populations. Three different primer sets were utilized in this study in order to assess the possibilities of primer binding site mutations. Population variation was measured in 460 U.S. Caucasian, 445 African American, 336 Hispanic, and 202 Asian samples along with mutation rates from almost 400 father–son pairs. In addition, the 10 genomic DNA components in NIST Standard Reference Material SRM 2391b were sequenced and found to exhibit a variety of additional base changes, insertions, and deletions outside of the SE33 repeat region.
The STR locus SE33, sometimes referred to as ACTBP2, is a core locus currently used with the German national DNA database and has been incorporated into several STR kits due to its highly polymorphic nature. Over 120 different alleles, spanning a size range of more than 100bp, have been reported to-date [1].
2. Materials and methods
A total of 1443 samples from U.S. populations groups that were self-identified as Caucasian (460), African American (445), Hispanic (336), and Asian (202) were examined with three different SE33 primer sets. The sources of these samples and additional studies on them have been previously reported [2], [3]. SE33 primers used in this study were part of the prototype PowerPlex® ESX 17 and ESI 17 Systems as well as the PowerPlex® ES Monoplex System, SE33 (JOE) available from Promega Corporation (Madison, WI). Manufacturer recommendations were followed for all PCR amplifications. PCR products were amplified on a GeneAmp 9700 (Applied Biosystems, Foster City, CA), separated on a 36cm array with an ABI 3130xl Genetic Analyzer (Applied Biosystems) using POP4 polymer, and genotyped using manufacturer supplied allelic ladders and bins and panels (Promega) on GeneMapperID v3.2 software (Applied Biosystems). DNA sequencing of NIST SRM 2391b components was performed using BigDye v3.1 chemistry (Applied Biosystems) and the following primers: 5′-CGTCTGTAATTCCAGCTCCTAGGGAG-3′ and 5′-AAGGACAAGGTTCTGTGCTCGCT-3′.
3. Results and discussion
As noted in Table 1, a total of 58 distinguishable SE33 alleles were observed, which is twice the number of alleles seen in the next most variable STR locus FGA.
Table 1.
Number of distinguishable alleles observed in 1443 samples with 16 STR loci examined using the PowerPlex ESX 17 and PowerPlex ESI 17 STR kits.
SE33
FGA
D21S11
D12S391
D18S51
D1S1656
D19S433
D2S441
58
29
28
24
23
17
16
15
D2S1338
D10S1248
D22S1045
D3S1358
D8S1179
vWA
D16S539
TH01
13
12
11
11
11
11
9
8
The only other published evaluation of SE33 allele frequencies in U.S. population groups was performed by Reid et al. [4] and involved examination of 200 Blacks, 200 Caucasians, and 200 Hispanics with the PowerPlex ES Monoplex primers. Fairly similar allele frequencies were observed between our study (Table 2) and the Reid et al. [4] information (data not shown). Two SE33 mutations were observed out of 391 father–son sample pairs providing a mutation rate of ∼0.5%. These single-step mutation results (20→21 and 15→14) were confirmed with multiple SE33 primer sets.
Table 2.
SE33 allele frequencies observed in 1443 U.S. population samples (2886 total alleles). The most common alleles in each group are indicated in bold font.
Allele
Total
Populations (%)
Total
Populations (%)
Total
Populations (%)
#
%
AfAm
Asian
Cauc
Hisp
Allele
#
%
AfAm
Asian
Cauc
Hisp
Allele
#
%
AfAm
Asian
Cauc
Hisp
6.3
1
0.1
0.1
7
1
0.1
0.1
8
1
0.1
0.1
10.2
1
0.1
0.1
11
1
0.1
0.1
11.2
2
0.1
0.2
12
11
0.4
0.3
0.5
0.4
12.2
4
0.1
0.2
0.3
13
31
1.1
1.1
1.5
1.0
13.2
9
0.3
1.0
14
85
2.9
5.1
0.2
2.5
2.4
14.2
10
0.3
0.4
0.4
0.3
15
102
3.5
3.9
1.2
3.9
3.9
15.2
8
0.3
0.3
0.7
16
144
5.0
4.8
4.7
4
6.7
16.2
5
0.2
0.3
0.1
0.1
16.3
2
0.1
0.3
17
205
7.1
9.3
4.0
6.2
7.3
17.2
1
0.1
0.1
17.3
5
0.2
0.1
0.2
0.3
18
268
9.3
12.1
5.0
7.2
11.0
18.3
1
0.1
0.1
19
249
8.6
12.2
6.2
6.6
8.0
19.2
8
0.3
0.2
0.4
0.4
20
216
7.5
10.9
9.2
5.4
4.8
20.2
20
0.7
0.3
1.2
1.1
0.3
21
108
3.7
4.6
6.7
2.4
2.7
21.2
48
1.7
1.1
1.7
2.4
1.3
22
42
1.5
1.3
1.7
1.5
1.3
22.2
65
2.3
0.4
3.2
3.8
1.9
23
12
0.4
0.6
1.0
0.2
0.1
23.2
91
3.2
2.2
4.2
4.3
2.1
24
1
0.1
0.1
24.2
74
2.6
1.3
6.2
2.2
2.5
25.2
110
3.8
2.7
6.9
4
3.1
26
1
0.1
0.1
26.2
163
5.6
6.1
5.2
4.3
7.1
27
1
0.1
0.1
27.2
225
7.8
4.3
10.4
9.5
8.6
27.3
2
0.1
0.3
28
2
0.1
0.1
0.2
28.2
180
6.2
4.4
7.9
7.4
6.1
28.3
2
0.1
0.1
0.1
29
1
0.1
0.2
29.2
147
5.1
2.7
5.7
6.3
6.3
29.3
1
0.1
0.2
30
1
0.1
0.1
30.2
111
3.8
1.6
3.2
5.8
4.6
31
3
0.1
0.1
0.2
31.2
52
1.8
1.5
2.5
2.2
1.3
32
1
0.1
0.1
32.2
25
0.9
0.4
0.7
1.3
0.9
33
2
0.1
0.1
0.1
33.2
11
0.4
0.3
0.5
0.4
34
9
0.3
0.3
0.7
34.2
1
0.1
0.1
35
1
0.1
0.1
36
2
0.1
0.2
51.7
67.2
40.3
43.2
50.0
48
31.9
59.2
56.1
48.3
0.5
0.2
0.2
0.4
1.0
Six discordant calls were observed between the three SE33 primer sets tested. All but one of the null alleles arose during amplification with the SE33 monoplex primer set, which is also used in the PowerPlex ES and AmpFlSTR SEfiler kits [5]. In one instance, allele 25.2 failed to be amplified with the prototype PowerPlex ESX 17 primers due to a primer binding site mutation (a C→T transition 75bp downstream of the repeat that impacts the reverse primer). While primer binding sites have been reported in previous SE33 concordance studies [6], [7], these new PowerPlex ESX 17 and PowerPlex ESI 17 primers sets appear to produce a lower level of null alleles.
Conflict of interest
None for NIST; Promega employees work for company supplying the kits.
Acknowledgments
Some of the DNA samples used in this study were kindly provided by Tom Reid of DNA Diagnostics Center and previously extracted and quantified by Jan Redman and Amy Decker from our group at NIST. This work was funded in part through interagency agreement 2008-DN-R-121 between the National Institute of Justice and the NIST Office of Law Enforcements. Points of view in this document are those of the authors and do not necessarily represent the official position or policies of the U.S. Department of Justice. Commercial equipment, instruments, and materials are identified in order to specify experimental procedures as completely as possible. In no case does such identification imply a recommendation or endorsement by the National Institute of Standards and Technology nor does it imply that any of the materials, instruments or equipment identified are necessarily the best available for the purpose.
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aNational Institute of Standards and Technology, Gaithersburg, MD 20899-8312, USA
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